Characterization of isolated acrosomal matrices from hamster spermatozoa

The acrosomal matrix of hamster spermatozoa was enriched and characterized. Acrosomal matrices were released from spermatozoa with shaking in a pH 5.2 buffer containing Triton X-100 and protease inhibitors, and enriched on a glass-bead column. Phase-contrast microscopy indicated that 70-80% of the acrosomal matrices were released from the spermatozoa and only minor contamination from sperm heads was detected. Transmission electron microscopy confirmed the low level of contamination in the preparation and revealed a bilaminar structure similar but not identical to that of guinea-pig acrosomal matrix. One- and two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the acrosomal matrix to be a complex structure enriched for several polypeptides. Proteinase activity was demonstrated by gelatin-SDS-PAGE. The major activity corresponded to bands of relative molecular masses (Mr) of 56,000, 51,000 and 48,000 with two minor bands of Mr 30,000 and 28,000. The lectin Pisum sativum agglutinin (PSA) bound to the anterior head of spermatozoa and isolated acrosomal matrix as judged by fluorescence microscopy using FITC-PSA. Western blots of spermatozoa and acrosomal matrices followed by overlay with biotinylated PSA indicated that there are at least two PSA-binding glycoproteins of Mr 60,000 and 72,000.     

A Rapid Purification of L-Glutamic Acid Decarboxylase from the Brain of the Locust Schistocerca gregaria

L-Glutamic acid decarboxylase (GAD; EC 4.1.1.15) was purified to apparent homogeneity from the brain of the locust Schistocerca gregaria using a combination of chromatofocusing (Mono P) and gel filtration (Superose 12) media. The homogeneity of the enzyme preparation was established by native polyacrylamide gel electrophoresis (PAGE) with silver staining. The molecular weight of the purified enzyme was estimated from native gradient gel electrophoresis and gel filtration chromatography to be 97,000 +/- 4,000 and 93,000 +/- 5,000, respectively. When analysed by sodium dodecyl sulphate-PAGE, the enzyme was found to be composed of two distinct subunits of Mr 51,000 +/- 1,000 and 44,000 +/- 1,500. Tryptic peptide maps of iodinated preparations of these two subunits showed considerable homology, suggesting that the native enzyme is a dimer of closely related subunits. The purified enzyme had a pH optimum of 7.0-7.4 in 100 mM potassium phosphate buffer and an apparent Km for glutamate of 5.0 mM. The enzyme was strongly inhibited by the carbonyl-trapping reagent aminooxyacetic acid with an I50 value of 0.2 microM.     

Purification and Characterization of a Sperm Motility Inhibiting Factor from Caprine Epididymal Plasma

Several studies have been reported on the occurrence of sperm motility inhibiting factors in the male reproductive fluids of different mammalian species, but these proteins have not been adequately purified and characterized. A novel sperm motility inhibiting factor (MIF-II) has been purified from caprine epididymal plasma (EP) by Hydroxylapatite gel adsorption chromatography, DEAE-Cellulose ion-exchange chromatography and chromatofocusing. The MIF-II has been purified to apparent homogeneity and the molecular weight estimated by Sephacryl S-300 gel filtration is 160 kDa. MIF-II is a dimeric protein, made up of two subunits each having a molecular mass of 80 kDa as shown by SDS-PAGE. The isoelectric point of MIF-II is 5.1 as determined by chromatofocusing and isoelectric focusing. It is a heat labile protein and maximal active at the pH 6.9 to 7.5. The sperm motility inhibiting protein factor at 2 µg/ml (12.5 nM) level showed maximal motility-inhibiting activity. The observation that the epididymal plasma factor lowered the intracellular cAMP level of spermatozoa in a concentration-dependent manner suggests that it may block the motility of caprine cauda spermatozoa by interfering the cAMP dependent motility function. The results revealed that the purified protein factor has the potential of sperm motility inhibition and may serve as a vaginal contraceptive. The antibody raised against the MIF-II has the potential for enhancement of forward motility of cauda-spermatozoa. This antibody may thus be useful for solving some of the problems of male infertility due to low sperm motility.     

Isotypes of protein kinase C in bovine sperm.

Protein kinase C is present in bovine epididymal sperm. The enzyme was partially purified by gel filtration on Sephacryl S-300. The Ca2+/phosphatidylserine-dependent histone phosphotransferase activity elutes from the gel filtration column in a manner corresponding to a Mr approximately 80 kDa. The activity peak also corresponds with [3H]phorbol 12,13-dibutyrate binding activity. Immunoblot analysis of the partially purified enzyme with isozyme-specific monoclonal antibodies revealed the presence of alpha-, beta-, and gamma-subspecies of protein kinase C. Indirect immunofluorescence showed that the antibodies against alpha-, beta-, and gamma-subspecies produced prominent staining of the postacrosomal region of the sperm head. In addition, beta-subspecies antibodies produced minor staining of the midpiece and gamma-subspecies antibodies produced a minor staining of the acrosomal region.     

Effects of seminal vesicle fluid components on sperm motility in the house mouse

Fluid obtained by stripping dissected seminal vesicles was mixed with phosphate-buffered saline and the soluble proteins were separated by gel filtration on BioRad P150 into 4 fractions. Fractions were collected and concentrated using an Amicon ultrafiltration system using YM2 membranes with a molecular weight cut-off of 1000. Epididymal sperm suspensions were incubated in medium containing one of the 4 fractions or 1 mg BSA/ml, or no added protein. After incubation for 2 h the motility of the spermatozoa in each suspension was assessed by a videomicrographic procedure. Two aspects of motility, velocity and the shape of the swimming path, were monitored. The results indicate that the seminal vesicles produce at least three factors that influence sperm motility. Fraction 3 (Mr 12,000-24,000) was detrimental to motility; after incubation for 2 h almost all the spermatozoa were immotile. Fractions 2 (Mr 25,000-40,000) and 4 (Mr 7000-12,000) both influenced the shape of the swimming path: spermatozoa incubated in Fraction 2 had straighter trajectories while those incubated in Fraction 4 showed more progressive paths with less side-to-side movement of the head about the path. These effects of factors from the seminal vesicle fluid on sperm motility may influence the way in which the spermatozoa move in the female reproductive tract and could help to explain why removal of the seminal vesicles reduces fertility in the mouse.     

The isolation and characterization of specific 3-methylcholanthrene-binding proteins from rat liver cytosol

The major proteins to which 3-methylcholanthrene specifically binds have been purified over 480-fold with a 45% yield compared to a rat liver 100,000g supernate. The procedure involved a batch ion-exchange technique together with hydrophobic gel filtration and chromatofocusing chromatography. The multiple, specific 3-methylcholanthrene-binding proteins obtained from this protocol had apparent isoelectric points of pH 6.3, 6.0, 5.7, and 5.5 on elution from a chromatofocusing column. They all shared a common sedimentation coefficient as determined by sucrose gradient analysis of 4.4 S. Gel filtration on Sephadex G-75 gave a common Stokes radius of 27 A. An analysis of these chromatofocusing peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed those which eluted at pH 6.3 and 6.0 to contain two major protein bands of Mr 32,000 and 34,000, together with several contaminating proteins. In contrast, the peaks from chromatofocusing which eluted at pH 5.7 and 5.5 contained three major proteins of Mr 40,000, 25,000, and 14,000. The specific binding capability of these chromatofocusing peaks was found to be unstable to temperatures of -30 degrees C and below. Competition studies showed that these proteins were not steroid receptors, and that only polycyclic aromatic hydrocarbons which could induce cytochrome P-450c were able to displace 3-methylcholanthrene from the binding site. A marked preference was noted for polycyclic aromatic hydrocarbons with four to five benzene rings arranged in a nonlinear fashion, suggesting the stereochemical requirements of the protein binding site. The stability of the noncovalent interaction between the proteins and 3-methylcholanthrene was in the range of pH 7 to 9.     

Effect of preincubation of cryopreserved porcine epididymal sperm

Preincubation of spermatozoa is important for capacitation and successful fertilization in vitro. The effects of preincubation time on frozen-thawed boar epididymal spermatozoa as measured by sperm motility, acrosomal integrity and fertilization ability in vitro were examined. Epididymal spermatozoa were collected from three Large White boars and frozen. The thawed spermatozoa were preincubated for 0, 15, 30, 60 and 120 min. Their motility was evaluated by a sperm motility analyzer and then the sperm motility indexes (SMIs) were calculated. The status of their acrosomal integrity was evaluated by triple-staining. Then, their fertilization ability was examined by in vitro fertilization (IVF) using porcine oocytes matured in vitro. SMIs of spermatozoa and the incidences of acrosome-intact live spermatozoa from the three boars were high (21-39 for SMI and 50-61% for acrosome-intact live spermatozoa) just after thawing, but both decreased as the duration of preincubation was prolonged (2-10 and 23-40%, respectively). The incidences of sperm penetration were high (61-89% of inseminated oocytes) when the sperm were preincubated for 0-60 min. However, sperm penetration decreased as the preincubation period was prolonged to 120 min. The degree of this decrease differed depending upon the boar from which the spermatozoa were obtained (10-72%). When the two parameters, sperm motility and acrosomal integrity, were analyzed statistically, the latter parameter rather than the former one showed a significant effect on penetration ability in vitro after each duration of preincubation. These results suggest that preincubation of frozen-thawed boar epididymal spermatozoa is not required for IVF and also that the maintenance of acrosomal integrity in unreacted status, rather than the maintenance of sperm motility, is important for fertilization ability after thawing and during preincubation of boar epididymal spermatozoa.     

Baculovirus expressed C-terminal fragment of bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 inhibits ZP3-mediated induction of acrosomal exocytosis

Zona pellucida glycoprotein-3 (ZP3) has been postulated as the primary sperm receptor in various mammalian species including bonnet monkey (Macaca radiata). However, information on the domain responsible for its binding to spermatozoa is inadequate. In the present study, bonnet monkey ZP3 (bmZP3), corresponding to amino acid (aa) residues 223-348 [bmZP3(223-348)] has been cloned and expressed using baculovirus expression system. SDS-PAGE and Western blot analysis of the purified renatured recombinant protein revealed it as a closely spaced doublet of approximately 25 kDa. Lectin-binding studies documented the presence of both O- as well as N-linked glycans. The biotinylated r-bmZP3(223-348) binds to the acrosomal region of the capacitated spermatozoa but fails to bind to the acrosome-reacted spermatozoa as investigated by immunofluorescence studies. In ELISA, nonbiotinylated r-bmZP3(223-348) and baculovirus expressed r-bmZP3, devoid of signal sequence and transmembrane-like domain [r-bmZP3(23-348)] competitively inhibit its binding to the capacitated spermatozoa. Interestingly, binding of biotinylated r-bmZP3(23-348) to the capacitated sperm is also inhibited by nonbiotinylated r-bmZP3(223-348). In contrast to r-bmZP3(23-348), r-bmZP3(223-348) failed to induce acrosomal exocytosis in the capacitated sperm. Interestingly, it competitively inhibits the acrosomal exocytosis induced by r-bmZP3(23-348). These studies, for the first time, identify a domain of ZP3 capable of binding to capacitated spermatozoa and inhibiting ZP3-mediated induction of acrosomal exocytosis furthering our understanding of mammalian fertilization.     

Calmodulin-stimulated protein kinase activity from rat pancreas

Previous work from our laboratory has demonstrated that neurohumoral stimulation of the exocrine pancreas is associated with the phosphorylation of the Mr 29,000 ribosomal protein S6. In a cell-free system using pancreatic postmicrosomal supernatant as the kinase donor, we found that the following co-factors stimulate the phosphorylation of the Mr 29,000 ribosomal protein: calcium with calmodulin, calcium with phosphatidyl serine, and cAMP. These findings suggest that the pancreas contains a calcium-calmodulin-dependent protein kinase (CaM-PK) that can phosphorylate the Mr 29,000 ribosomal protein. A CaM-PK activity was partially purified sequentially by ion exchange, gel filtration, and calmodulin-affinity chromatography. Phosphorylation of the Mr 29,000 ribosomal protein by the partially purified CaM-PK was dependent on the presence of both calcium and calmodulin and not on the other co- factors. The CaM-PK fraction contained a phosphoprotein of Mr 51,000 whose phosphorylation was also dependent on calcium and calmodulin. When 125I-calmodulin-binding proteins from the CaM-PK fraction were identified using electrophoretic transfers of SDS-polyacrylamide gels, a single Mr 51,000 protein was labeled. The preparation enriched in CaM- PK activity contained an Mr 51,000 protein that underwent phosphorylation in a calcium-calmodulin-dependent manner and an Mr 51,000 calmodulin-binding protein. It is therefore possible that the CaM-PK may comprise a calmodulin-binding phosphoprotein component of Mr 51,000.     

Regulation of proacrosin conversion in isolated guinea pig sperm acrosomal apical segments

Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proacrosin has been identified in extracts of intact guinea spermatozoa as a major silver staining band which reacted immunologically with antibodies made against purified proacrosin from guinea pig testis. Proacrosin exhibited an approximate Mr of 50,000 and was rapidly converted to an Mr 45,000 protein following induction of the acrosome reaction with 2.0 mM CaCl2 and 1 micrograms/ml A23187. Apical segments isolated at pH 6.0 from guinea pig spermatozoa also contained a major silver staining band of Mr 50,000 which cross-reacted with antibodies to guinea pig testis proacrosin. Subcellular fractionation of spermatozoa indicated that proacrosin remained in the particulate fraction of homogenized spermatozoa and was enriched within the isolated acrosomal apical segment. When apical segments isolated at pH 6.0 were incubated at pH 7.5, proacrosin was rapidly converted to the Mr 45,000 form observed in spermatozoa undergoing the acrosome reaction. The conversion process in isolated apical segments was inhibited by leupeptin and was accelerated in the presence of calcium, magnesium, and manganese. Zinc completely inhibited the conversion of proacrosin to the Mr 45,000 protein. Neither proacrosin nor the Mr 45,000 protein were released into the supernatant fluid during the incubation of apical segments at pH 7.5. Furthermore, the proteins were resistant to solubilization by 150 mM NaCl and 1% Triton X-100 but were solubilized by treatment of apical segments with 1 M NaCl. These results provide evidence as to the identity and subcellular distribution of proacrosin in intact guinea pig sperm prior to zymogen conversion and suggest that isolated apical segments exhibit a subset of the exocytotic reactions leading to completion of the acrosome reaction.     

Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human TPX1 and is a member of the cysteine-rich secretory protein (CRISP) family

We have cloned and sequenced cDNAs encoding autoantigen 1 (AA1), a testis-specific protein and the major autoantigen of the guinea pig sperm acrosome. The cDNA predicts a precursor protein of 244 amino acids including a 21 amino acid hydrophobic, secretory signal sequence. The mature polypeptide is predicted to have a molecular mass of 24,891 Daltons which agrees with the experimentally determined molecular weight of 25,000. Consistent with previous studies demonstrating that AA1 is not a glycoprotein, the predicted amino acid sequence contained no canonical sites for N-linked glycosylation. Comparison with other sequences showed that AA1 is the guinea pig homologue of the testis-specific protein Tpx-1 in mice and TPX1 in humans. AA1 also showed significant amino acid sequence homology with other cysteine-rich secretory proteins (CRISP's): rat and mouse acidic epididymal glycoproteins (AEG; also known as proteins D/E in rats) and helothermine, a toxin from the Mexican beaded lizard. In addition, AA1 had a lesser degree of homology with antigen 5 (vespid wasp venom), PR-1 (a plant pathogenesis related protein), and GliPR (a protein identified in human gliomas). Northern analysis of RNA from purified guinea pig spermatogenic cells showed that a 1.5 kb message was first detected in pachytene spermatocytes, was strongest in round spermatids, and was detected at a low level in condensing spermatids. Immunoblot analysis and metabolic labeling data of AA1 in spermatogenic cells showed that the protein was synthesized as early as the pachytene spermatocyte stage of spermatogenesis. Thus, the patterns of AA1 mRNA and protein expression during spermatogenesis are similar to the expression of other acrosomal mRNAs and proteins that are first detected meiotically. © 1996 Wiley-Liss, Inc.     

Identification of sea urchin sperm adenylate cyclase

Calmodulin (CaM) affinity chromatography of a detergent extract of sea urchin sperm yielded approximately 20 major proteins. One of these proteins, of Mr 190,000, was purified and used to immunize rabbits. After absorption with living sperm, the serum reacted monospecifically on one- and two-dimensional Western immunoblots with the Mr 190,000 protein. The anti-190-kD serum inhibited 94% of the adenylate cyclase (AC) activity of the CaM eluate. An immunoaffinity column removed 95% of the AC activity, and the purified (but inactive) Mr 190,000 protein was eluted from the column. The antiserum also inhibited 23% of the activity of bovine brain CaM-sensitive AC and 90% of the activity of horse sperm CaM-sensitive AC. These data support the hypothesis that the Mr 190,000 protein is sea urchin sperm AC. Although this AC bound to CaM, it was not possible to demonstrate directly a Ca2+ or CaM sensitivity. However, two CaM antagonists, calmidazolium and chlorpromazine, both inhibited AC activity, and the inhibition was released by added CaM, suggesting the possibility of regulation of this AC by CaM. Indirect immunofluorescence showed the Mr 190,000 protein to be highly concentrated on only the proximal half of the sea urchin sperm flagellum. This asymmetric localization of AC may be important to its function in flagellar motility. This is the first report of the identification of an AC from animal spermatozoa.     

First attempt to characterize 3H-fucose acrosomal label in spermatozoa

Summary Acrosomes of rabbit spermatozoa were labelled by tritiated fucose introduced into the cells during spermatogenesis. The labelling was analysed simultaneously by autoradiography and biochemically. In compact intact acrosomes the labelling was confined strictly to the acrosomal region of the sperm head. In swollen and detached acrosomes the autoradiographic grains were associated mostly with the acrosomal cap. Only in some cells a small proportion of radioactivity was detected to be associated with denuded sperm heads. In acrosomal extracts a considerable share of radioactivity coincided with gel filtration fractions showing esterase activity (BAEE-Nalpha-benzoyl-l-arginine ethyl ester splitting), akin to that exhibited by acrosin.     

Calcium-induced modification of the acrosomal matrix in digitonin-permeabilized guinea pig spermatozoa

In this report we describe and partially characterize a preparation of digitonin-permeabilized guinea pig spermatozoa that undergo a rapid and synchronous modification of the acrosomal matrix in response to calcium. Permeabilization of cauda epididymal spermatozoa by digitonin was monitored by using adenylate cyclase activity as an indicator. Spermatozoa (5 x 10(7) cells) treated with 0.005% digitonin for 15 s exhibited maximal adenylate cyclase activity but generally retained their structural morphology, as examined by phase-contrast and transmission electron microscopy. The ratio fo cell number to detergent concentration was the critical factor for determining both the efficiency of permeabilization and the maintenance of structural integrity. When permeabilized spermatozoa were treated with 2 mM CaCl2, the cells underwent a rapid and synchronous modification of the acrosomal matrix (AM). As observed by phase-contrast microscopy, the response to CaCl2 was characterized by events that occurred in the following temporal sequence: disruption of the sperm rouleaux, the loss of refractility by the apical segment of the sperm acrosome, and detachment of the apical segment from the spermatozoa. Transmission electron microscopy indicated that the loss of refractility from the sperm apical segment was coincident with a calcium-induced dispersion of the AM. Analysis of the proteins released during this response, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that a specific subset of sperm proteins was released from the spermatozoa, including a major = staining, 45,000 Mr protein apparently generated from a higher molecular weight precursor during the acrosome reaction.     

Acrosome biogenesis begins during meiosis: evidence from the synthesis and distribution of an acrosomal glycoprotein, acrogranin, during guinea pig spermatogenesis.

The biogenesis of the sperm-specific organelle, the acrosome, was investigated using an acrosomal glycoprotein as a marker of development. This component, which we have named acrogranin, was purified from an acid extract of guinea pig testes by standard chromatographic procedures. The molecular weight of reduced acrogranin was determined to be 67,000 by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunization of female rabbits with purified acrogranin produced an antiserum that recognized a single protein with Mr = 67,000 in an acid extract of guinea pig testes. By indirect immunofluorescence, acrogranin was found only in the acrosome of mature sperm. In haploid spermatids, acrogranin was localized in the developing acrosome and, weakly, in the cytoplasm. Acrogranin was also detected in the cytoplasm and juxtanuclear region in putative proacrosomal granules of meiotic cells (pachytene spermatocytes). Detergent extracts from different purified germ cell populations contained only the Mr = 67,000 form of acrogranin, but sperm extracts had four lower Mr immunoreactive forms not present in the testicular extracts. By two-dimensional gel electrophoresis, acrogranin was found to be an acidic glycoprotein. Analysis of glycosylated and trifluoromethanesulfonic acid-deglycosylated acrogranin indicated that the antibody recognized polypeptide determinants. After highly enriched germ cell populations were labeled overnight with [35S]methionine and extracted with detergent, anti-acrogranin immunoprecipitated a single protein of Mr = 67,000. The synthesis of acrogranin by pachytene spermatocytes and round spermatids was similar, but the synthesis of the glycoprotein by condensing spermatids was markedly reduced. These studies demonstrate that acrosome biogenesis, as determined by the synthesis of a specific acrosomal component, begins during meiosis and continues through the early stages of spermiogenesis.     

Characterization of an acrosomal matrix protein in hamster and bovine spermatids and spermatozoa.

Experiments have been carried out characterizing an Mr 22,000 protein present in the acrosomes of hamster and bull spermatozoa. The Mr 22,000 protein is resistant to solubilization in detergent solutions containing high or low salt and has a pI of -5.2. With various lectins, the protein from hamster sperm was shown to be sparingly glycosylated with N-acetylglucosamine, mannose, and galactose while that from the bull demonstrated a slight reactivity for galactose. Using a specific monoclonal antibody (MAB 4/18), the Mr 22,000 polypeptide has been localized exclusively to the acrosomes of mature testicular and epididymal hamster and bovine sperm. Acrosomal components of differentiating bovine and hamster spermatids in tissue sections did not react with the monoclonal antibody, although the protein was present in immunoblots of round spermatids. In bovine sperm, MAB 4/18-staining at the ultrastructural level with immunogold-labeled second antibody was present as a reticulum throughout the acrosomal cap and as punctate aggregates in the equatorial segment. In hamster sperm, MAB 4/18-reactivity was present along the periphery of the acrosome in conjunction with matrix components (M1 and M2), as well as along the inner acrosomal membrane. These observations indicate that the acrosomes of bovine and hamster sperm possess an immunologically related Mr 22,000 protein and suggest that differences in MAB 4/18-staining of spermatids and spermatozoa is a result of epitope modification and/or a change in accessibility of the epitope to the antibody probe during the course of spermiogenesis. Based on its localization and solubility properties, we suggest that the Mr 22,000 protein, in conjunction with other polypeptides, forms a structural framework to maintain acrosomal shape and/or compartmentalize acrosomal contents.     

First attempt to characterize 3H-fucose acrosomal label in spermatoza

Acrosomes of rabbit spermatozoa were labelled by tritiated fucose introduced into the cells during spermatogenesis. The labelling was analysed simultaneously by autoradiography and biochemically. In compact intact acrosomes the labelling was confined strictly to the acrosomal region of the sperm head. In swollen and detached acrosomes the autoradiographic grains were associated mostly with the acrosomal cap. Only in some cells a small proportion of radioactivity was detected to be associated with denuded sperm heads. In acrosomal extracts a considerable share of radioactivity coincided with gel filtration fractions showing esterase activity (BAEE-N-alpha-benzoyl-L-agrinine ethyl ester splitting), akin to that exhibited by acrosin.     

The status of acrosomal caps of hamster spermatozoa immediately before fertilization in vivo

Adult female golden hamsters were induced to superovulate. When they were mated several hours prior to ovulation or artificially inseminated about the time of ovulation, nearly 100% of their eggs were subsequently fertilized monospermically. During the progression of fertilization when the eggs were still surrounded by compact cumulus oophorus, the contents of the ampullary region of the oviducts were collected and spermatozoa moving in the ampullary fluid, within the cumulus and on/in the zonae pellucidae of unfertilized eggs, were examined by light and electron microscopy to evaluate the status of their acrosomal caps. Most spermatozoa swimming in the ampullary fluid had apparently intact acrosomal caps, while the vast majority moving within the cumulus had distinctly modified acrosomal caps. Most spermatozoa that had passed through the cumulus and reached the zona surfaces had remnants of their acrosomal caps (“acrosomal ghosts”). When the ghosts were present around the sperm heads on the zona, the heads pivoted about a point roughly corresponding to the places where the ghosts were located. The ghosts seemed to firmly attach to the zona surfaces, then were split open by the sperm heads and left behind as the sperm heads advanced into the zona. A few spermatozoa on the zona surfaces had no acrosomal ghosts (at least not detectable by light microscopy). In this case, the sperm head pivoted about either the inner acrosomal membrane or the equatorial segment of the acrosome. In no instance were spermatozoa with intact acrosomal caps found on zona surfaces. We infer from these observations that most spermatozoa in vivo initiate their acrosome reactions while they are advancing through the cumulus. When they arrive at the zona surfaces, acrosomal ghosts are generally present on the sperm heads. These ghosts appear to hold sperm heads to zona surfaces as well as to restrict the direction of advancement of sperm head through the zona. In a minority of cases, ghostless spermatozoa reach the zona surfaces. As these spermatozoa appear to be able to penetrate the zona successfully, structures other than the acrosomal ghost (ie, the inner acrosomal membrane and the plasma membrane over the equatorial segment of the acrosome) may also attach to zona surfaces before spermatozoa penetrate into the zona.     

Scanning electron-microscopical and other observations of sperm fertilization reactions in Limulus polyphemus L. (Merostomata: Xiphosura).

Sperm fertilization reactions of Limulus polyphemus were examined by scanning electron and/or light microscopy. The following were considered: sperm motility, attachment of sperm to egg, acrosome reaction, and penetration of the acrosomal filament. The spermatozoa after semination are non-motile and become active only in close proximity to a defined region surrounding the egg. Egg materials diffusing into this region induce sperm motility and stimulate large numbers of spermatozoa to move towards the egg surface. Each sperm initially attaches by the apical tip and undergoes the acrosome reaction which causes a more permanent secondary attachment by the adhesion of acrosomal contents to the egg surface. The acrosome reaction also initiates the penetration of the acrosomal filament through the egg envelope, an event occurring in 70-80% of the attached spermatozoa (about 10(6). Shortly after this penetration, a secondary reaction occurs which involves a spiralling of the flagellum and an incorporation into the sperm body of the flagellar fibrous components, which then become closely apposed to the sperm nucleus. These sperm fertilization reactions were performed or initiated with 0-34 M CaCl2 in whole eggs, egg sections, excised egg envelopes and/or the outer basement lamina of the egg envelope. The Limulus fertilization system is very valuable since sperm reactions can be examined biochemically, which may lead to a better understanding of the chemical mechanisms involved in sperm-egg interactions in all animal species.     

Phosphatidylcholine and phosphatidylinositol-specific phospholipases C of bull and rabbit spermatozoa.

Acrosomal reaction is an essential prerequisite to fertilization. The changes in lipid composition of sperm membranes cause fusion of the plasma and outer acrosomal membranes that results in the exocytosis of acrosomal contents. We report that both bull and rabbit spermatozoa contain a phosphatidylcholine-specific phospholipase C (PC-PLC) that hydrolyzes L-alpha-dipalmitoyl-(choline-methyl-14C-153.0 Ci/mmol and a phosphatidylinositol-specific phospholipase C (PI-PLC) that hydrolyzes L-alpha-(Myo-Inositol-2-3H (N)-5.2 Ci mmol. PI-PLC from bull sperm acrosome has been purified 568 x fold with a specific activity 6.25 +/- 0.6 nmol/min/mg protein, km 0.004 mM, and Vmax 12 nmol/min/mg protein. Both enzymes had optimum at pH 7.5. The activity of PC-PLC remained unaffected by varying concentrations of Ca2+, whereas PI-PLC activity was significantly increased. The bulk of PI-PLC was found to be associated with inner acrosomal membrane of bull and rabbit sperm, while PC-PLC was found in the outer acrosomal membranes in the bull sperm and the plasma membrane of the rabbit sperm. Both enzymes are compartmentalized in sperm cell.